Patent Application
20090093010
The present invention relates to methods and kits for monitoring kidney function and detecting kidney dysfunction.
Renal insufficiency is associated with many pathological conditions. Decreased kidney function can be indicative of renal transplant rejection, as well as other organ rejection. Acute tubular necrosis, transient hypertension and preeclampsia during pregnancy, and chronic glomerular diseases can also result in increased proteinuria and enzymuria indicative of decreased kidney function. Furthermore, nephrotoxicity can be secondary to environmental toxic agents such as lead, cadmium, mercury and perchlorethilene as well as pharmaceutical drug toxicity. Therefore, accurate assessment of kidney function has application and significant prognostic value in the clinic.
Although short and long-term kidney allograft survival has improved substantially from 1988-1996 (1), this trend did not continue from 1995-2000 (2). Specifically, despite a continuous decrease in reported acute clinical rejection rates within the first year post-transplant in the latter period, allograft recipient survival actually diminished (2). This was attributed to “a higher proportion of acute rejection episodes which have not resolved with full functional recovery in recent years” (2), but it may also be due to undetected—and therefore untreated—rejection episodes (i.e. subclinical rejection) which harm the allograft over time.
Both immunological factors and non-immunological factors (for example calcineurin-inhibitor (CNI)-toxicity, hypertension, and recurrent disease) contribute to the continuous deterioration of allograft function, which is referred to as chronic allograft nephropathy (CAN) (3). Acute allograft rejection is the major immunological risk factor for developing CAN (4,5). However, there remains a consistent rate of late graft loss due to CAN with or without previous acute clinical rejection episodes suggesting the existence of subtle and ‘subclinical’ degrees of graft inflammation that are capable of progressing to CAN. Indeed while non-immunological factors may play a role, a recent analysis found that immunological factors were strong correlates of declining graft function beyond 6 months (6).
Currently about 50% of renal allografts are lost due to patient death with a functioning graft. These patients die mainly from cardiovascular diseases and malignancies (7,8). While this finding may reflect the overall increasing age of renal transplant recipients, it may also be influenced by more potent immunosuppressive regimens used to date, which increase cardiovascular risk factors (e.g. hypertension, hypercholesterinemia) and malignancy development (9). In addition, new emerging opportunistic viral infections such as polyomavirus BK-type nephropathy (10,11) underscore the observation that over-immunosuppression may have increased in current years. With this concern in mind, there has been a recent interest in the implementation of strategies that reduce the net immunosuppression delivered to the patient by avoidance, minimization, withdrawal or substitution drug protocols (12). The problem with such strategies, however, is that there has been to date no way other than a renal allograft biopsy of ascertaining whether the graft is free of rejection, and several attempts at reducing immunosuppression have been followed by acute rejection episodes.
Therefore, the individualization of the immunosuppressive therapy tailored to the needs of every patient at every time point is a major goal. To achieve this, tools to monitor the rejection process in the allograft are mandatory. However, this is complicated by the complex and often redundant biology of allograft rejection.
At present, the diagnosis of acute rejection can only be made by renal allograft biopsy, which provides information about the type (humoral vs. cellular) and the severity of rejection (tubulointerstitial vs. vascular) that can be used to select the appropriate anti-rejection therapy. Sometimes it is ‘practical’ in a clinical setting to assume rejection by excluding other possibilities for graft dysfunction and to treat rejection. Nevertheless, most kidney transplant centres perform an allograft biopsy when rejection is a concern and allograft function (measured by serum creatinine) has deteriorated by more than 20-30% from baseline.
However, studies by the Winnipeg Transplant Group have demonstrated that the serum creatinine is an insensitive method for the early detection of renal allograft pathology. Indeed, the histologic criteria for acute rejection are present in 3-45% of protocol biopsies of renal allografts with stable function (‘subclinical rejection’) (42,43,44,45,46). The pathogenic potential of subclinical rejection was demonstrated in a randomized study in which the treatment of early subclinical rejection with corticosteroids improved both early and late outcomes (43). Specifically, there was a decrease in early (months 2-3) as well as late (months 7-12) clinical rejection episodes, a decrease in the chronic tubulointerstitial pathological score at 6 months, and a lower serum creatinine at 24 months in those patients randomized to treatment. Finally, similar to acute pathology, the Winnipeg Transplant Group reported that early chronic allograft pathology, detectable only by a 6-month protocol biopsy (i.e. graft function was stable), is predictive of both a subsequent decline in allograft function and time to graft failure (47,48). These data suggest that early detection and treatment of subclinical inflammation may be required to decrease the incidence of CAN.
With the advent of new immunosuppressive agents it is becoming apparent that a limitation to the ‘gold standard’ (i.e. renal biopsy) is the extent of heterogeneity of inflammation within the allograft resulting in sampling error (49). To overcome this obstacle one could take additional cores, use larger biopsy needles or perform more frequent protocol biopsies. However, clearly this is restricted by patient risk for complications that limits the frequency with which they can be performed, not to mention the associated cost. An alternative is to further increase the baseline immunosuppression for all patients, but this carries the known risks of infection in the short-term, and of drug toxicity and malignancy in the long-term. Therefore, in order to detect and eventually prevent these early pathogenic lesions, it is important to develop non-invasive approaches that sample the entire graft and can be performed repeatedly.
Non-invasive monitoring of the immune response directed at the kidney allograft is constrained to examine cells or proteins from the peripheral blood or urine. Strategies have broadly taken one of two approaches (50). The first takes advantage of donor-recipient MHC disparity, the central target of the alloimmune response, to design donor antigen specific assays. The second strategy is to assess global changes in immune system components of the recipient. As will be discussed below each approach offers distinct advantages and disadvantages. Independent of the strategy however, any clinical assay should be conducted easily with small volumes of blood or urine and be able to be repeated frequently.
These approaches have largely employed donor cells as targets for either recipient T-cells or sera containing antibody targeting donor-MHC. To date the most successful by far has been the ‘cross-match’ assay examining pre-transplant sera for donor specific alloantibodies that target MHC molecules on the surface of donor T- or B-cells (51). In contrast to antibody assays, donor-specific T-cell assays have not proven to be as predictive (50). Tests have included limiting dilution assays (LDA), trans-vivo delayed type hypersensitivity (DTH) assays, enzyme-linked immunospot (ELISPOT) assay, flow cytometry based detection of cytokines, and tetramer staining. Like the antibody ‘cross-match’ assay, the LDA and ELISPOT assays have been successful in detecting pre-transplant donor-specific T-cell memory that predict risk for early acute rejection (52). However, their utility to monitor for acute rejection post-transplant has been rather limited (53). While highly specific for donor antigens, the main disadvantages of these assays are: [I] the need for a repository of donor cells (limits frequency of testing possible), [II] the need for cell expansion (time consuming and labour intensive), [III] reproducibility is poor, [IV] complex interpretation, [V] low sensitivity, [VI] in the case of tetramers requires availability of a diverse panel with a number of potential donor-recipient disparities, and [VII] in the case of trans-vivo DTH the need for a large number of animals (50).
To date antigen non-specific assay development (via immuno-phenotyping for immune cell activation markers, cytokine excretion, or mRNA analysis) has largely been limited to known inflammatory programs that are associated with clinical rejection (50,54,55,56,57,58,59,60). However, it is unclear whether these assays will reliably detect the more subtle (subclinical) forms of acute and/or chronic rejection. The Winnipeg Transplant group attempted to develop non-invasive markers correlating clinical and subclinical rejection with flow based detection of CD69 up-regulation on circulating T-cells (i.e. an early T-cell activation marker that was found in the biopsy infiltrate of acute clinical and subclinical rejection). In this study, CD69 expression tended to correlate with acute allograft inflammation, however, it was also up-regulated when asymptomatic cytomegalovirus (CMV) viremia was present in the blood (61). This study highlighted the difficulty in using antigen non-specific biomarkers; it is difficult to ensure specificity since activation of immune markers in blood can reflect inflammation generated through multiple pathways (i.e. rejection versus infection) and occurring at multiple sites within the patient. In addition, T-cells in the circulation may not necessarily be representative of their abundance within the graft (62). The same problems apply also to studies measuring serum proteins secreted by immune cells (e.g. IL-2, IL-6, IFN-γ). Although statistically significant differences have been found in patients with or without acute rejection, the overlap of the two populations was often substantial (58,59) resulting in either many ‘false positives’ or many ‘false negatives’ for a selected cut-off.
Urine as a specimen for immune monitoring offers some potential advantages compared to serum, because [I] it is in direct contact with the main target of rejection (tubular epithelial cells), [II] it may represent the whole kidney allograft, and [III] it may be less confounded by systemic inflammation. However, urine can be very heterogeneous concerning the amount of cells, the concentration of proteins and the pH. One group used mRNA measurement of granzyme B, perforin and CD103 in urinary lymphocytes to predict acute renal allograft rejection (56,57), others measured cytokines (59) or chemokines (60). Yet again, the major problem in these studies was the insufficient sensitivity and specificity, which limits the clinical usefulness of such assays. The unsatisfactory performances could partially be explained by the rather loose definition of ‘no-rejection’ in these studies, which was mostly based on stable allograft function without further support by allograft histology.
Excreted enzymes and low molecular weight proteins have been used as markers of nephron toxicity including transplant rejection. β2-microglobulin is a low molecular weight protein that has been extensively studied for its association with transplant rejection, drug toxicity, and renal proximal tubular function.
β2-microglobulin consists of 99 amino acids with one disulfide bridge and has a molecular weight of 11,731 Da (Swiss-Prot: P61769). It is non-covalently bound to the class I major histocompatibility antigen and found on the cell surface of all nucleated cells. Production of β2-microglobulin is known to be between 150 to 250 mg/day in healthy individuals, whereas an increase is observed in some lymphoproliferative and autoimmune diseases (reviewed in (16,17)). β2-microglobulin is shed from the cell surface and circulates in serum, 98% as a free form (18). Most free β2-microglobulin is filtered by the glomeruli and ≧99.9% reabsorbed by proximal tubular cells (17), where it is thought to be degraded into peptides/amino acids by lysosomes before reuptake into the circulation. Therefore, in healthy individuals with normal proximal tubular function <0.2 mg/L β2-microglobulin is excreted in urine (13,14,15).
Due to these properties, increased intact urinary β2-microglobulin has been considered an ideal biomarker for proximal tubular dysfunction. However, it was soon realized that intact β2-microglobulin values measured by immunoassays decreased significantly over time in urine with a pH<6, suggesting proteolytic activity, which leads to cleaved β2-microglobulin forms that were not detectable by available immunoassays (19,20). This fact largely limited the usefulness of measuring intact urinary β2-microglobulin. Although adding alkali to urine post void can prevent degradation ex vivo, most degradation occurs in vivo, as urine is normally stored in the bladder for at least 2-3 hours prior to voiding. Therefore, the only way to accurately measure intact urinary β2-microglobulin is to give patients alkali (e.g. sodium bicarbonate) systemically to ensure a urine pH≧6 or to analyse only urine samples with pH≧6 (17). By following these steps, the potential of intact urinary β2-microglobulin as a marker for proximal tubular injury has been demonstrated by completely separating patients with lower urinary tract infection from those with pyelonephritis without overlap (15). However, the need for administration of alkali prior to urine analysis and the restriction of using only urine samples with pH≧6 made the measurement of intact urinary β2-microglobulin unattractive for routine clinical use.
Therefore, due to the limitations and problems associated with existing invasive and non-invasive methods of monitoring transplant rejection as well as the need for accurate assessment of kidney function in a plethora of conditions, it would be desirable to identify new non-invasive biomarkers of kidney function and methods of detecting and monitoring these markers in patients.
The present inventors have devised a high-throughput method for analyzing the proteome of samples from animals and correlating the protein profile to disease or disorder induced kidney dysfunction. Kidney dysfunction is an indicator of diseases and disorders including but not limited to drug toxicity, heavy metal poisoning, renal tubular damage and other kidney disease, transplant disease including transplant rejection, and systemic diseases such as diabetes, lupus, and rheumatoid arthritis. The inventors have found that the presence of a distinct urinary protein profile correlates with kidney dysfunction.
The inventors have further shown that the distinct protein profile identified from individuals with kidney dysfunction is comprised of cleaved β2-microglobulin protein fragments. These β2-microglobulin protein fragments are useful as diagnostic and prognostic biomarkers of kidney dysfunction. Furthermore, assays for the presence of β2-microglobulin protein fragments may be used to monitor kidney function over time.
The methods of the invention are advantageously non-invasive. The β2-microglobulin protein fragments of the invention are associated with kidney dysfunction and can be detected in urine samples. This allows for frequent measurement, which may further improve clinical outcome by better individualization of therapeutic interventions.
Accordingly, in one embodiment, the present invention provides a method of detecting kidney dysfunction in an animal comprising:
(a) testing a sample from the animal for the presence of β2-microglobulin protein fragments, wherein the presence of one or more β2-microglobulin protein fragments when compared to a control sample indicates that the animal has kidney dysfunction.
In an embodiment of the invention, the β2-microglobulin protein fragments are one or more than one of the fragments selected from the group consisting of I1-Y63 (SEQ ID NO:2), I1-F62 (SEQ ID NO:3), I1-S61 (SEQ ID NO:4), E69-M99 (SEQ ID NO:5), F70-M99 (SEQ ID NO:6), Y66-M99 (SEQ ID NO:7), Y67-M99 (SEQ ID NO:8) and T68-M99 (SEQ ID NO:9).
In a preferred embodiment of the invention, the sample being tested is urine.
The inventors have also shown that the presence of β2-microglobulin protein fragments indicates that a patient has a transplant related disease, and that the presence of β2-microglobulin protein fragments can be a prognostic indicator of transplant rejection. In particular, patients undergoing subclinical transplant rejection can be identified, permitting immunosuppressive therapies to be tailored to early events in transplant rejection.
Accordingly, in one embodiment, the present invention provides a method of detecting kidney transplant related disease in an animal that has received a transplant comprising:
(a) testing a sample from the animal for the presence of β2-microglobulin protein fragments, wherein the presence of one or more β2-microglobulin protein fragments when compared to a sample from a normal animal indicates that the animal has a kidney transplant related disease.
In a preferred embodiment, a method of the invention is used to detect transplant rejection. In another preferred embodiment, a method of the invention is used to detect subclinical rejection. In yet another preferred embodiment the sample being tested is urine.
Diseases and disorders may induce chronic kidney dysfunction or acute kidney dysfunction. Chronic kidney dysfunction may be interrupted by periods of acute kidney dysfunction. It is necessary to monitor kidney function over time referenced to the individual protein profile over time. Furthermore, repeated testing is desirable to monitor therapeutic efficacy following a particular treatment or course of therapy.
Accordingly, the present invention also provides a method of monitoring kidney function in an animal comprising:
(a) testing a sample from the animal to determine the level of β2-microglobulin protein fragments;
(b) repeating step (a) at a later point in time and comparing the result obtained in step (a) with the result obtained in step (b) wherein a difference in the level of β2-microglobulin protein fragments is indicative of a change in kidney function.
In an embodiment of the invention, the β2-microglobulin protein fragments are one or more than one of the fragments selected from the group consisting of I1-Y63 (SEQ ID NO:2), I1-F62 (SEQ ID NO:3), I1-S61 (SEQ ID NO:4), E69-M99 (SEQ ID NO:5), F70-M99 (SEQ ID NO:6), Y66-M99 (SEQ ID NO:7), Y67-M99 (SEQ ID NO:8) and T68-M99 (SEQ ID NO:9).
In a preferred embodiment of the invention, the sample being tested is urine.
The present inventors have also shown that protease activity is elevated in the urine of patients with kidney dysfunction.
Accordingly, the present invention further provides a method of detecting kidney dysfunction in an animal comprising:
(a) testing a urine sample from the animal for protease activity, wherein increased protease activity when compared to a control sample indicates that the animal has kidney dysfunction.
In one embodiment, a method of the invention is used to detect transplant rejection. In another embodiment, a method of the invention is used to detect kidney dysfunction induced by a systemic disease selected from the group consisting of diabetes, lupus, or rheumatoid arthritis. In another embodiment a method of the invention is used to detect diabetes induced kidney dysfunction. In yet another embodiment, a method of the invention is used to detect kidney dysfunction induced by drug toxicity. In a preferred embodiment the sample being tested is urine.
The present invention also provides biomarkers that can be used in the detection and prognosis of kidney transplant related disease and which are useful for assessing transplant function and health.
Accordingly, in one embodiment the invention provides a biomarker for detecting kidney dysfunction in an animal comprising at least one β2-microglobulin protein fragment.
Moreover, the present invention provides kits for detecting kidney dysfunction in an animal comprising (i) reagents for conducting a method according to a method of the invention and (ii) instructions for its use.
In a preferred embodiment a kit of the invention is used to detect transplant rejection. In another preferred embodiment, the transplant is a kidney transplant. In yet another preferred embodiment the sample being tested is urine.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The invention will now be described in relation to the drawings in which:
Assessment of kidney function is a prognostic indicator of disease progression and can be used to determine adequacy of treatment. As described above, the available methods of assessing kidney function are inadequate for detecting early disease progression.
Renal insufficiency is associated with many pathological conditions. Decreased kidney function can be indicative of renal transplant rejection, as well as other organ rejection. Acute tubular necrosis, transient hypertension and preeclampsia during pregnancy, and chronic glomerular diseases can also result in increased proteinuria and enzymuria indicative of decreased kidney function (119). Diabetes and cancer can also impact kidney function. Furthermore, nephrotoxicity can be secondary to environmental toxic agents such as lead, cadmium, mercury and perchlorethilene as well as pharmaceutical drug toxicity (119). Hence accurate assessment of kidney function has application and significant prognostic value in the clinic.
The present inventors have provided non-invasive methods for the monitoring of kidney function and detection of kidney dysfunction and kidney transplant related disease, based on the presence of β2-microglobulin protein fragments.
Accordingly, in one embodiment, the present invention provides a method of detecting kidney dysfunction in an animal comprising:
(a) testing a sample from the animal for the presence of β2-microglobulin protein fragments, wherein the presence of one or more β2-microglobulin protein fragments when compared to a control sample indicates that the animal has kidney dysfunction.
The term “sample from the animal” as used herein means any sample including, but not limited to, biological fluids, tissue extracts, freshly harvested cells, and lysates of cells which have been incubated in cell cultures. In a preferred embodiment, the sample is urine.
As used herein the phrase “β2-microglobulin protein fragments” or “fragments of the β2-microglobulin protein” means a fragment or portion of the full length β2-microglobulin protein and includes polymorphic versions of amino acid sequences of all of the known β2-microglobulin molecules and precursor molecules, including those deposited in GenBank under accession number CAA23830 or those referred to in Suggs et al. Proc. Natl. Acad. Sci. U.S.A. 78 (11), 6613-6617 (1981), as well as modified versions including those referred to in Momoi et al., Clin Chim Acta. 1995 May 15; 236(2):135-44, and any variants, analogs, derivatives or portions thereof that are useful in detecting transplant related disease.
The term “animal” as used herein includes all members of the animal kingdom, including humans. Preferably, the animal is a human.
In a preferred embodiment, β2-microglobulin protein fragments are selected from the group consisting of I1-Y63 (SEQ ID NO:2), I1-F62 (SEQ ID NO:3), I1-S61 (SEQ ID NO:4), E69-M99 (SEQ ID NO:5), F70-M99 (SEQ ID NO:6), Y66-M99 (SEQ ID NO:7), Y67-M99 (SEQ ID NO:8) and T68-M99 (SEQ ID NO:9) (letters indicate single letter amino acid code; numbers indicate position of amino acids in full-length β2-microglobulin protein sequence). β2-microglobulin protein giving rise to the β2-microglobulin protein fragments may be cleaved at one or more of the following sites: tyrosine-63 (Y-63), leucine-65 (L65), phenylalanine-62 (F62), and serine-61 (S61). The major distinct protein fragments resulting from these cleavages may have the approximate molecular weights of 7358 Da, 7195 Da or 7048 Da. Additionally the β2-microglobulin long chain may be cleaved at one or more of the following sites: phenylalanine-22 (F-22), asparagine-24 (N24) and cysteine-25 (C-25). Fragments resulting from these cleavages may or may not be detectable. The β2-microglobulin protein may or may not also be cleaved in its short chain at lysine-75 (K75), glutamic acid-74 (E74), threonine-73 (T73), proline-72 (P-72), threonine-71 (T71), phenylalanine-70 (F70) and/or glutamic acid-69 (E69). Two fragments resulting from these cleavages may have the approximate molecular weight of 3737 Da and 3608 Da.
The term “control sample” includes any sample that can be used to establish a base or normal level, and may include samples taken from healthy animals or samples mimicking physiological fluid.
As used herein “kidney dysfunction” means abnormal tubular function resulting in the loss of proteins into the urine that are normally absent from the urine.
As used herein “non-invasive” refers to a method whereby the sample to be tested can be obtained without biopsy. Preferably “non-invasive” refers to a method whereby the sample to be tested can be obtained without puncturing the skin of the animal.
As used herein “protein profile” means the group of protein fragments obtained from a sample and is used interchangeably with “distinct protein profile” or “protein profile pattern” or “protein pattern”. The protein profile can indicate whether the animal has a kidney dysfunction related disease or disorder such as a transplant rejection.
Diseases and disorders may induce chronic kidney dysfunction or acute kidney dysfunction. Chronic kidney dysfunction may be interrupted by periods of acute kidney dysfunction. It is necessary to monitor kidney function over time referenced to the individual protein profile over time. Furthermore, repeated testing is desirable to monitor therapeutic efficacy following a particular treatment or course of therapy. Therefore, the methods of the invention are also used to monitor the adequacy of therapeutic interventions.
Accordingly, the present invention also provides a method of monitoring kidney function in an animal comprising:
(a) testing a sample from the animal to determine the level of β2-microglobulin protein fragments;
(b) repeating step (a) at a later point in time and comparing the result obtained in step (a) with the result obtained in step (b) wherein a difference in the level of β2-microglobulin protein fragments is indicative of a change in kidney function.
There are four main categories responsible for allograft injury: (i) rejection episodes, (ii) drug-toxicity (i.e. calcineurin-inhibitors (CI)), (iii) specific diseases (e.g. polyomavirus type BK-nephropathy, recurrent disease in the allograft), and (iv) disease accelerating factors (e.g. hypertension, diabetes) (41). Acute clinical rejection is the major risk factor for allograft failure (4), but even rejection episodes without allograft dysfunction as measured by serum creatinine (i.e. subclinical rejection detected by protocol biopsies) can lead to chronic allograft nephropathy (116,117). Moreover, CI-nephrotoxicity was reported in >50% of protocol biopsies performed after the second year post-transplant (42). However, protocol biopsies have not gained widespread acceptance due to their associated costs, inconvenience and morbidity. Non-invasive biomarkers in serum or urine, which can be measured frequently, may guide the clinical decision to perform an allograft biopsy. Indeed, sensitive, non-invasive biomarkers of tissue injury may allow the clinician to determine its cause (i.e. by allograft biopsy) before irreversible damage has occurred. Furthermore, the response to therapeutic interventions can be followed by frequent measurement of such biomarkers (118).
Post-transplant immune monitoring of renal transplant recipients is currently based on the integrated information gathered from the allograft function (i.e. serum creatinine), the risk profile of a patient (e.g. number of MHC-mismatches, presensitization), the clinical course (e.g. prior rejections) and ultimately the allograft biopsy results. While these tools have proved to be invaluable for adjusting the immunosuppressive therapy, they still have major shortcomings as described above.
Immune monitoring with non-invasive markers, which allows for frequent measurement, may further improve the clinical outcome of the allograft recipient by better individualization of immunosuppressive therapy. Specifically, this includes reduction of immunosuppressive therapy for patients inferred to be free of rejection by the non-invasive test, as well as increasing immunosuppressive therapy before tissue damage occurs and the rejection process becomes obvious (i.e. worsening allograft function). Non-invasive, antigen-specific tests are mostly labour intensive, expensive and required donor cells (with the exception of tetramer-staining), and do not lend themselves to high-throughput analysis in busy clinical settings. Non-antigen specific tests are cheaper and have high-throughput capabilities, but they often lack sensitivity and specificity for allograft rejection. As urine is [I] in direct contact with the main target of rejection (tubular epithelial cells), [II] may represent the whole kidney allograft, and [III] may also be less confounded by systemic inflammatory processes, non-invasive biomarkers in urine may have a higher sensitivity and specificity than serum biomarkers. Finally, proteins, as the effector molecules, may be more informative and specific for the rejection process than gene transcription products (i.e. mRNA).
Accordingly, in one embodiment, the present invention provides a method of detecting kidney transplant related disease in an animal that has received a transplant comprising:
(a) testing a sample from the animal for the presence of β2-microglobulin protein fragments, wherein the presence of one or more β2-microglobulin protein fragments when compared to a sample from a normal animal indicates that the animal has a kidney transplant related disease.
In a preferred embodiment a method of the invention is used to detect transplant rejection.
As used herein “transplant” means a tissue or organ transplanted from a donor of the same or of a different species and includes allografts and xenografts. Furthermore “transplant” includes solid organ transplants and kidney transplants.
As used herein “transplant related disease” comprises illnesses and conditions affecting the transplant such as transplant rejection, acute allograft rejection, subclinical rejection episodes, interstitial fibrosis, fibrous intimal thickening of arteries, and calcineurin-inhibitor toxicity. When referring to a kidney transplant, “transplant related disease” further comprises tubular stress and injury, tubular atrophy, glomerulosclerosis, polyomavirus type BK-nephropathy (BK-NP), chronic allograft nephropathy (CAN), and pyelonephritis (PN).
As used herein “transplant rejection” means the presence of an immunological inflammatory response in the transplant. With respect to kidney transplants, it means the presence of an immunological inflammatory response in the kidney transplant that is targeting the tubulointerstitial compartment of the kidney. When the transplant related disease is transplant rejection, the distinct protein profile identified following analysis of urine samples is sometimes referred to as a “rejection pattern”.
Currently about 50% of kidney transplants are lost due to patient death with a functioning graft. The potent immunosuppressive regimens used to date increase cardiovascular risk factors such as hypertension and hypercholeserinemia and increase malignancy development (9), which may contribute to transplant patient death rates. Over-immunosuppression may also increase the risk for developing opportunistic infections, which may further complicate transplant management. The invention provides a non-invasive method of detecting a transplant related disease that can be performed repeatedly and analyzed quickly. One of the advantages of the current invention is that the non-invasive nature of the methods permits repeated testing and better individualization of immunosuppressive therapies.
The sample tested may be serum, blood, urine or tissue. Urine as a specimen for immune monitoring in renal transplants offers some potential advantages compared to serum. It is in direct contact with the main target of rejection and may represent the whole kidney transplant. Furthermore it may be less confounded by systemic inflammation. In a preferred embodiment of the invention, the animal sample tested is urine. In a further preferred embodiment, the urine sample is a mid-stream urine sample.
In a further aspect of the present invention, a non-invasive method for the detection and monitoring of transplant health and for the early detection and monitoring of transplant rejection is provided.
Accordingly, in one embodiment the invention provides a method of monitoring transplant health in an animal comprising:
(a) testing a sample from the animal to determine the level of β2-microglobulin protein fragments;
(b) repeating step (a) at a later point in time and comparing the result obtained in step (a) with the result obtained in step (b) wherein a difference in the level of β2-microglobulin protein fragments is indicative of a change in transplant health.
As used herein “transplant health” means an assessment of organ function that is compared to a clinically defined normal organ function (i.e. based on creatinine levels) or “normal” transplant function.
The inventors have shown that the presence of a protein profile indicative of transplant related disease, in particular the presence of β2-microglobulin protein fragments, precedes other measures of clinical rejection (i.e. defined change in serum creatinine levels). The invention permits, in one embodiment, the identification of individuals undergoing subclinical rejection. This allows for greater individualization of immunosuppressive therapies. Studies have demonstrated the pathogenic potential of subclinical rejection and early treatment can improve both early and late outcomes (43). Monitoring transplant health is advantageous since it allows for the reduction of immunosuppressive therapy for patients inferred to be free of rejection. It further permits for immunosuppressive therapies to be augmented or altered before tissue damage occurs and the rejection process becomes obvious (i.e. worsening allograft function). One of the advantages of the current invention is that the non-invasive nature of the methods permits repeated testing and better individualization of immunosuppressive therapies.
As used herein “subclinical rejection” means stable transplant function but wherein the transplant exhibits some histologic criteria of acute rejection.
In a preferred embodiment a method of the invention is used to detect or monitor sub-clinical transplant rejection. In a further preferred embodiment, the transplant is a kidney transplant. In another preferred embodiment the sample being tested is urine.
A protein profile can be assessed by one of several methods including, but not limited to, gel electrophoresis including 2D gel electrophoresis; chromatography including liquid chromatography; protein microarray; isotope coded affinity tags; hydrolytic labeling; and mass spectrometry including SELDI-TOF-MS. In a preferred embodiment the protein profile is detected using a SELDI-TOF-MS platform.
SELDI-TOF-MS provides many advantages for the protein profiling of urine samples. A small volume of sample (i.e. 5-10 μL) is needed for each analysis and many samples can be analyzed quickly. This permits high-throughput profiling of many samples. Furthermore, washing steps are easily incorporated and this has the advantage of removing most of the salts, which interfere with mass spectrometric analysis.
Other groups have used SELDI-TOF-MS to compare the protein profiles between different clinical outcomes, but required bioinformatic analysis to assign protein peaks to a specific outcome (98,99). In another study, Clarke et al. (98) reported differences in the urine profiles between rejection and stable transplants; however, Clarke et al.'s requirement of bioinformatics to do so may relate to the fact that their definition of ‘stable’ transplants was less stringent than that of the present inventors (i.e. based on serum creatinine alone). Interestingly, the protein peaks reported in the Clarke et al. paper as specific to rejection, are different from those found by the present inventors. This may be related to the different protein chip surfaces and experimental conditions that were utilized; but also, to the fact that Clarke et al. (98) failed to include any control populations (e.g. ATN, recurrent or de novo glomerulopathies, UTI, CMV) in the analysis, the importance of which is discussed below. In another study, Petricoin et al. (99) have used SELDI-TOF-MS to compare the protein profiles between different clinico-pathological diagnoses in cases of ovarian cancer, but also required bioinformatic analysis to assign peaks to specific outcomes. In the Petricoin et al. study the analysis involved serum samples which is clearly a more complex biological fluid than urine. Indeed, the urine-based proteomics has the advantage of excluding most of the serum proteins from the urine due to the size/charge selectivity of the glomerular basement membrane.
In a preferred embodiment, the protein profile detected using SELDI-TOF-MS is comprised of 1-3 Regions or clusters of one or more distinct protein fragments. In one embodiment Region 1 preferably consists of 5 distinct fragments. In another embodiment Region 2 preferably consists of 3 distinct fragments. In a further embodiment the distinct fragments are clustered in three regions, wherein Region 1 comprises 5 fragments; Region 2 consists of 3 fragments; and Region 3 consists of 5 fragments.
The present invention also provides quantitative assays for detecting protein fragments. These quantitative assays permit the detection of changes in concentration of intact protein, of protein fragments, and of intact protein and fragments, and may be immunological in nature. Immunological assays can be based on: (i) the detection of neoepitopes arising as a result of cleavage of intact protein or protein fragments; (ii) the determining of the ratio of binding of antibodies directed at different epitopes present on the whole molecule or fragments thereof, wherein the loss of epitopes (i.e. cleavage of intact protein or protein fragments) would cause a shift; or (iii) the appearance of fragments which could be captured and displayed using a range of different physical methods, for example polyacrylamide gel electrophoresis or mass spectrometry.
In a preferred embodiment a method of the invention is used to detect transplant rejection. In a further preferred embodiment, the transplant is a kidney transplant. In yet another preferred embodiment the sample being tested is urine.
The present invention also provides biomarkers that can be used in the detection and prognosis of kidney transplant related disease and which are useful for assessing transplant function and health.
Accordingly, in one embodiment the invention provides a biomarker for detecting kidney dysfunction in an animal comprising at least one β2-microglobulin protein fragment.
As used herein “biomarker” means at least one protein fragment that can be used for one or more of the following: to detect that an animal has a disease; to predict that an animal will develop a disease; to monitor the progression of a disease; or to monitor the effect of a treatment.
A biomarker may have various uses. An early intervention (or diagnostic) biomarker is used for early detection of disease to facilitate intervention. A prognostic biomarker is used to identify patients who may benefit from an intervention (63). Ideally, a biomarker has both, diagnostic and prognostic properties.
A diagnostic biomarker is described by its sensitivity, specificity and its receiver operating characteristics (ROC) curve. ROC-analysis allows finding the best cut-off value to assign the test result to be ‘positive’ or ‘negative’. For clinical decision-making, it is more important to know the positive (PPV; ‘true positives’) and negative predictive value (NPV; ‘true negatives’) than its sensitivity and specificity. This calculation then allows determination of how many ‘false positive’ and ‘false negative’ results the test produces. These numbers should be as low as possible, because they represent the patients that are wrongly assigned to have either a ‘positive’ or a ‘negative’ test. Besides the given and constant factors that affect sensitivity and the specificity of a diagnostic test, the prevalence of the target disease in the screened population largely influences the PPV, the NPV, the number of ‘false positives’ and the number of ‘false negatives’. Therefore, these values should always be calculated based on the ‘true prevalence’ of the disease in the screened population rather than from a selected population, which may over- or underestimate the ‘true prevalence’ and consequently lead to wrongly calculated PPV and NPV (64).
A prognostic biomarker should preferably ‘predict’ the outcome of a particular condition. Prediction requires the further criterion of showing that changes in the value have consequential changes in the outcome. Many prognostic biomarkers used to date only ‘correlate’ with an outcome (e.g. C-reactive protein and risk of acute myocardial infarction), fewer ‘predict’ (e.g. smoking and risk of lung cancer or acute myocardial infarction).
Serum β2-microglobulin protein levels have been found to increase in patients undergoing renal transplant rejection (Backman L et al. Transplantation 42: 368, 1986) and heart transplant (Erez E et al. J. Heart Lung Transplant 17: 538, 1998) and increased expression of β2-microglobulin has been observed in the bile ducts, hepatocytes and endothelial cells of patients undergoing liver transplant rejection (Hubscher S G et al J. Clin Pathol. 41: 1049). Urine β2-microglobulin levels have also been examined for a potential association with transplant rejection but the results have been conflicting. Prischl and colleagues reported that out of 100 episodes of clinical rejection, 50 had only a moderate increase in urine β2-microglobulin levels (Prischl F et al. Nephron 1989; 51(3):330-7) and others found no increase in urine β2-microglobulin during episodes of renal rejection (Steinhoff J et al. Clin Nephrol. 1991 June; 35(6):255-62). The inventors have found that fragments of β2-microglobulin detectable in urine can serve as biomarkers for transplant rejection.
In one embodiment a biomarker of the invention comprises at least one β2-microglobulin protein fragment which is selected from the group consisting of: I1-Y63 (SEQ ID NO:2), I1-F62 (SEQ ID NO:3), I1-S61 (SEQ ID NO:4), E69-M99 (SEQ ID NO:5), F70-M99 (SEQ ID NO:6), Y66-M99 (SEQ ID NO:7), Y67-M99 (SEQ ID NO:8) and T68-M99 (SEQ ID NO:9).
In a preferred embodiment a biomarker of the invention is used to detect transplant rejection. In another preferred embodiment, the transplant is a kidney transplant.
In several embodiments of the invention the methods involve the detection of β2-microglobulin protein fragments. In a preferred embodiment, β2-microglobulin protein fragments are detected using antibodies that specifically bind to β2-microglobulin protein fragments. Antibodies to β2-microglobulin protein fragments can readily be prepared by a person skilled in the art.
Antibodies to β2-microglobulin protein fragments may be prepared using techniques known in the art. For example, by using a peptide of a β2-microglobulin protein fragment, polyclonal antisera or monoclonal antibodies can be made using standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenic form of the peptide which elicits an antibody response in the mammal. Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art. For example, the protein or peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies. Following immunization, antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.
To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e.g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256, 495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al. Monoclonal Antibodies in Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96), and screening of combinatorial antibody libraries (Huse et al., Science 246, 1275 (1989)). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated.
The term “antibody” as used herein is intended to include fragments thereof which also specifically react with one or more β2-microglobulin protein fragments or sub-fragments thereof. Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above. For example, F(ab′)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments.
Chimeric antibody derivatives, i.e., antibody molecules that combine a non-human animal variable region and a human constant region, are also contemplated within the scope of the invention. Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions. Conventional methods may be, used to make chimeric antibodies containing the immunoglobulin variable region which recognizes the gene product of β2-microglobulin antigens of the invention (See, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81, 6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom patent GB 2177096B). It is expected that chimeric antibodies would be less immunogenic in a human subject than the corresponding non-chimeric antibody.
Monoclonal or chimeric antibodies specifically reactive with a protein of the invention as described herein can be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin. Such immunoglobulin molecules may be made by techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)), and PCT Publication WO92/06193 or EP 0239400). Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)
Specific antibodies, or antibody fragments, such as, but not limited to, single-chain Fv monoclonal antibodies reactive against β2-microglobulin protein fragments may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with peptides produced from the nucleic acid molecules of β2-microglobulin fragments. For example, complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al., Nature 341, 544-546: (1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)). Alternatively, a SCID-hu mouse, for example the model developed by Genpharm, can be used to produce antibodies or fragments thereof.
Antibodies specifically reactive with β2-microglobulin protein fragments, or derivatives, such as enzyme conjugates or labeled derivatives, may be used to detect β2-microglobulin protein fragments in various samples (e.g. biological materials). They may be used as diagnostic or prognostic reagents and they may be used to detect abnormalities in the level of protein expression, or abnormalities in the structure, and/or temporal, tissue, cellular, or subcellular location of β2-microglobulin protein fragments. In vitro immunoassays may also be used to assess or monitor the efficacy of particular therapies. The antibodies of the invention may also be used in vitro to determine the level of expression of a gene encoding β2-microglobulin protein fragments in cells genetically engineered to produce β2-microglobulin protein fragments.
The antibodies may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of β2-microglobulin protein fragments and the antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, and histochemical tests. The antibodies may be used to detect and quantify β2-microglobulin protein fragments in a sample in order to determine its role in transplant rejection and to diagnose transplant rejection.
In particular, the antibodies of the invention may be used in immunohistochemical analyses, for example, at the cellular and subcellular level, to detect one or more β2-microglobulin protein fragments, to localize it to particular cells and tissues, and to specific subcellular locations, and to quantitate the level of expression.
Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect β2-microglobulin protein fragments. Generally, an antibody of the invention may be labeled with a detectable substance and β2-microglobulin protein fragments may be localized in tissues and cells based upon the presence of the detectable substance. Examples of detectable substances include, but are not limited to, the following: radioisotopes (e.g., 3H, 14C, 35S, 125I, 131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), luminescent labels such as luminol; enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups (which can be detected by marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods), predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached via spacer arms of various lengths to reduce potential steric hindrance. Antibodies may also be coupled to electron dense substances, such as ferritin or colloidal gold, which are readily visualized by electron microscopy.
The antibody or sample may be immobilized on a carrier or solid support which is capable of immobilizing cells, antibodies etc. For example, the carrier or support may be nitrocellulose, or glass, polyacrylamides, gabbros, and magnetite. The support material may have any possible configuration including spherical (e.g. bead), cylindrical (e.g. inside surface of a test tube or well, or the external surface of a rod), or flat (e.g. sheet, test strip). Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against β2-microglobulin protein fragments. By way of example, if the antibody having specificity against β2-microglobulin protein fragments is a rabbit IgG antibody, the second antibody may be goat anti-rabbit gamma-globulin labeled with a detectable substance as described herein.
Where a radioactive label is used as a detectable substance, β2-microglobulin protein fragments may be localized by radioautography. The results of radioautography may be quantitated by determining the density of particles in the radioautographs by various optical methods, or by counting the grains.
Labeled antibodies against β2-microglobulin protein fragments may be used in identifying patients undergoing transplant rejection i.e. in imaging. Typically for in vivo applications, antibodies are labeled with radioactive labels (e.g. iodine-123, iodine-125, iodine-131, gallium-67, technetium-99, and indium-111). Labeled antibody preparations may be administered to a patient intravenously in an appropriate carrier at a time several hours to four days before the tissue is imaged. During this period unbound fractions are cleared from the patient and the only remaining antibodies are those associated with the transplant. The presence of the isotope is detected using a suitable gamma camera.
The β2-microglobulin protein fragments may also be detected using nucleic acid aptamers. Aptamers are macromolecules such as RNA or DNA that can bind a specific target such as a protein or protein fragment. The three-dimensional shape of the nucleic acid allows it to bind tightly to its target. Aptamers are highly specific and can distinguish between closely related molecules and may be useful for distinguishing between β2-microglobulin protein fragments and β2-microglobulin protein. In addition they exhibit high affinity for their target and can have affinities in the picomolar to nanomolar range for proteins. Aptamers can be modified to reduce their sensitivity to enzymatic degradation and may be immobilized on a solid carrier or support as similarly described above for antibodies.
The inventors have characterized the protease(s) involved in fragmenting β2-microglobulin. The inventors have confirmed earlier observations (111,112), that the cleavage/degradation of urinary β2-microglobulin requires a pH<6. The responsible enzymes belong to the aspartic protease family as only pepstatin could prevent β2-microglobulin cleavages at pH 5 and most aspartic proteases have their pH optimum in the acidic range. Recently, two members of the aspartic protease family have been detected in human urine (cathepsin D (113) and napsin A (114)). Both enzymes are primarily located in lysosomes and are involved in protein degradation. Cathepsin D is found in the kidney in the distal tubules and collecting ducts (109), whereas napsin A is mainly found in the proximal tubules (115,108). The degradation of intact urinary β2-microglobulin by cathepsin D has been demonstrated, however, only two of the reported cleavage sites found by N-terminal sequencing are consistent with the 26 found in this study (113). This suggests that other aspartic proteases are involved in cleavage of urinary β2-microglobulin.
Accordingly, in one embodiment, the present invention provides a method of detecting kidney dysfunction in an animal comprising:
(a) testing a urine sample from the animal for protease activity, wherein increased protease activity when compared to a control sample indicates that the animal has kidney dysfunction.
In a preferred embodiment, the urine sample from the animal is tested for aspartic protease activity.
In another preferred embodiment, the urine sample from the animal is tested for the activity of an aspartic protease selected from the group consisting of cathepsin D and napsin A.
The present invention also provides assays for detecting the activity of the protease(s) involved in fragmenting β2-microglobulin. These assays include assays to detect the cleavage of selected substrates (synthetic or native), for example peptide substrates bearing one or more known cleavage sites, utilizing a sample from a patient. Enzyme activity can be measured in a number of ways: (i) calorimetrically, (ii) by release of radioactive fragments, (iii) by conducting fragment analysis (gels, mass spectrometric), or (iv) immunologically, based upon the appearance or loss of reporter epitopes. Many of these methods of measurement and detection are well known in the art. Details of the specific assay would vary with the approach chosen.
The methods described herein may be performed by utilizing pre-packaged diagnostic kits comprising the necessary reagents to perform any of the methods of the invention.
Accordingly, in one embodiment the invention provides a kit for detecting transplant related disease in an animal comprising (i) reagents for conducting a method of the invention and (ii) instructions for its use.
The kits may include at least one specific nucleic acid or antibody described herein, which may be conveniently used, e.g., in clinical settings, to monitor kidney function, to detect kidney dysfunction, and to screen, monitor and diagnose transplant recipients for transplant health or the development of transplant related disease. For example, the nucleic acid may be an aptamer that interacts with a β2-microglobulin protein fragment. The kits may also include nucleic acid primers for amplifying nucleic acids encoding protein profile distinct protein fragments in the polymerase chain reaction. The kits can also include nucleotides, enzymes and buffers useful in the method of the invention as well as electrophoretic markers such as a 200 bp ladder. The kits can also include antibodies that specifically bind β2-microglobulin or fragments thereof, and secondary antibodies for detecting those primary antibodies. The kit will also include detailed instructions for carrying out the methods of the invention.
The following non-limiting examples are illustrative of the present invention.
All patient data (e.g. allograft function measured by serum creatinine, biopsies) and urine data were stored and managed in a central access database. From July 1997 to March 2003, 2400 serial mid-stream urine samples from 212 renal transplant patients were collected. These 212 patients underwent a total of 693 protocol or clinically indicated core needle allograft biopsies. All patient charts were reviewed and additional information extracted as needed. Biopsies were analysed by experienced renal pathologists, and scored according to the Banff 1997 classification (Table 3) (23). The acute Banff score determines acute interstitial (ai 0-3), tubular (at 0-3), vascular (av 0-3) and glomerular (ag 0-3) changes, whereas the chronic Banff score assesses chronic interstitial (ci 0-3), tubular (ct 0-3), vascular (cv 0-3) and glomerular (cg 0-3) changes. The individual scores are added to a total acute (a 0-12) and total chronic (c 0-12) score. A biopsy specimen was judged adequate, when ≧7 glomeruli and ≧1 vessel were available for analysis. All patients were treated with a triple immunosuppressive regimen consisting of calcineurin-inhibitor (cyclosporine or tacrolimus), prednisone and mycophenolate-mofetil or azathioprine.
[1] Normal control group: Consists of 28 healthy individuals (14 female and 14 male, age 20-50 years).
[2] Urinary tract infection (UTI) group: Consists of 5 females with an episode of a lower UTI, which was defined as requiring the clinical symptoms of a UTI, a leukocyte count in the urine sediment >40 per high power field and a positive bacterial culture (>108 colony forming units).
Second-morning urine from healthy men and women were collected in two different containers. The first 10-20 mL of urine collected was considered first-void urine, the following 50-80 mL mid-stream urine. Urines were centrifuged in a fixed angle centrifuge for 10 minutes at 2000 rpm (900 g), the supernatants were transferred into 2 mL cryo-tubes (Gordon Technologies Inc., Missisauga, ON) and stored at −80° C. until further analysis. All samples were obtained with informed consent and ethics approval of the University of Manitoba Institutional Review Board. For urine sediment analysis 10 mL of freshly collected urine was centrifuged for 10 minutes at 2000 rpm. The pellet was analyzed with a phase-contrast microscope at 400× magnification and is reported as cells per high power field (hpf).
All urine samples were stored non-centrifuged at −80° C. until further analysis. All transplanted patient and control group urine samples were obtained with informed consent and ethics approval by the University of Manitoba institutional review board.
Urine samples were thawed on ice, shortly vortexed and centrifuged for 5 minutes at 10000 rpm (to remove remaining cell particles). Two different ProteinChips were used for the analysis. They were prepared as follows:
[1] Normal phase chips (ProteinChip NP20; Ciphergen, Freemont, Calif.): Five μL of urine supernatant were applied in duplicate to the chip and incubated for 20 minutes in a humidity chamber. Spots were then washed three times with 5 μL HPLC-grade water and air-dried for 10 minutes.
[2] Hydrophobic chips (ProteinChip H4): Five μL of 50% acetonitrile in HPLC-grade water were applied to the spots for 5 minutes to activate the surface. This solution was removed and 5 μL urine supernatant were applied in duplicate to the chip and incubated for 20 minutes in a humidity chamber. Spots were washed twice with 5 μL 10% acetonitrile in HPLC-grade water and then once with 5 μL HPLC-grade water. Chips were air-dried for 10 minutes.
As matrices saturated α-cyano-4-hydroxycinnamic acid (CHCA: Ciphergen) and sinapinic acid (SPA: Ciphergen) were prepared in 50% acetonitrile/0.5% trifluoro-acetic acid (TFA) according to the manufacturer's instructions and 1 μL of matrix solution (35% CHCA unless otherwise specified) was applied to each spot and air-dried. Unless stated otherwise, chips were read with the following SELDI-TOF-MS instrument (ProteinChip Reader II: Ciphergen) settings in the positive ion mode: Laser intensity 230; detector sensitivity 6; detector voltage 1800 V; positions 20 to 80 were read with an increment of 5 (resulting in 13 different sampling positions); sixteen laser shots were collected on each position (total shots collected and averaged: 208/sample); eight warming shots were fired at each position, which were not included in the collection; the acquired mass range was from a mass-over-charge (m/z) ratio of 0 to 80000; lag time focus of 900 ns. Calibration was done externally with a mixture of 4 proteins with masses ranging from 2 to 16 kDa. After baseline subtraction, peak labeling was performed by the ProteinChip Software (Version 3.1) for peaks with a signal-to-noise (S/N) ratio of ≧3 in the m/z range from 2000-80000. For some comparisons and presentations spectra were normalized according to the total ion current.
A urine sample with the rejection pattern proteins was dialysed with 7 kD cut-off dialysis cassettes (Slide-A-Lyzer, Pierce, Rockford, Ill.) against 50 mmol/L MES pH 6 and 50 mmol/L Tris pH 8, respectively. Cation-exchange (CM HyperD, Ciphergen) and anion-exchange (Q HyperD, Ciphergen) beads were washed three times for 20 minutes with 1 mL 50 mmol/L MES pH 6 or 50 mmol/L Tris pH 8, respectively. The pH 6 fraction was incubated on CM-beads for 2 h in a ratio of 5 μL beads per 1 mL urine. The supernatant was transferred to a separate tube. After washing the CM-beads twice with two bead-volumes 50 mmol/L MES pH 6 for 15 minutes, proteins were eluted with increasing concentrations of KCl in 50 mmol/L MES pH 6 (two bead-volumes for 30 minutes each). The supernatant and the eluted fractions were checked for the presence or absence of the rejection pattern proteins by SELDI-TOF-MS. The pH 8 fraction was incubated on Q-beads for 2 h in a ratio of 5 μL beads per 1 mL urine. The supernatant was transferred to a separate tube. After washing the Q-beads twice with two bead-volumes 50 mmol/L Tris pH 8 for 15 minutes, proteins were eluted with increasing concentrations of NaCl in 50 mmol/L Tris pH 8 (two bead-volumes for 30 minutes each). The supernatant and the elution fractions were checked for the presence or absence of the rejection pattern proteins by SELDI-TOF-MS.
Purification of Rejection Pattern Proteins with Cation Exchange (CM) Beads and Reverse-Phase High-Pressure Liquid Chromatography (RP-HPLC)
Fifteen mL of urine sample with the rejection pattern proteins was dialysed with 6-8 kD cut-off dialysis tube membrane (Spectra/Por, Spectrum Laboratories, Rancho Dominguez, Calif.) against 50 mmol/L MES pH 6.2. Dialysed urine was transferred into 1.5 mL siliconized tubes (Fisherbrand) and previously washed CM-beads (see above) were added in a ratio of 5 μL beads per 1 mL urine. After 2 h incubation the supernatant was transferred to a separate tube and the CM-beads were washed twice with two bead-volumes 50 mmol/L MES pH 6.2 for 15 minutes. Proteins were eluted with two beadvolumes 200 mmol/L KCl in 50 mmol/L MES pH 6.2. Those fractions containing the rejection pattern proteins were lyophilized and resuspended in a 5 times smaller volume of HPLC-grade water.
Further purification was done by RP-HPLC using an Agilent 1100 Series with a C4 column (Zorbax SB-C4, 5 μm, 0.5×150 mm; Agilent Technologies, Paulo Alto, Calif.). Five μL of concentrated sample was applied and eluted using a 1.6% acetonitrile increment per minute in 0.1% TFA during the first 17 minutes, followed by a 0.3% increment per minute for 24 minutes and a 16% increment per minute for the last 4 minutes at a flow rate of 20 μL/minute. Peak fractions containing the rejection pattern proteins were pooled, lyophilised and resuspended in 50 mmol/L ammonium bicarbonate for in solution digestion. The purification process was monitored with SELDI-TOF-MS using H4 chips.
Concentrated and purified protein (from about 10 mL starting material) was reduced with 10 mM DDT for 30 minutes at 57.5° C., alkylated with 50 mM iodoacetamide for 30 minutes in the dark, then dialysed against 50 mmol/L ammonium bicarbonate, and finally digested with 100 ng trypsin (sequencing-grade modified trypsin, Promega) over night at 37° C. Peptides were lyophilised, resuspended in 5 μL 0.1% TFA, and subjected to RP-HPLC separation using an Agilent 1100 Series system with a C18 column (Vydac 218 TP C18, 5 μm, 0.15×150 mm). Peptides were eluted with a linear gradient of 1.3% acetonitrile increment per minute in 0.1% TFA during 35 minutes and a 10% increment for the last 5 minutes. The column effluent (4 μl/min) was mixed online with 2,5-dihydroxybenzoic acid (0.16 g/ml, Sigma-Aldrich) matrix solution (0.5 μl/min) and deposited by a small computer-controlled robot onto a movable MALDI target at one-minute intervals. Forty such fractions were collected over a total period of 40 minutes. The spots on the target were analyzed individually, both by single mass spectrometry (MS) and by tandem mass spectrometry (MS/MS) in the Manitoba/Sciex prototype quadrupole/time-of-flight mass spectrometer (QqTOF) (81). In this instrument, ions are produced by irradiation of the target with photon pulses from a 20-Hz nitrogen laser (Laser Science) with 300 mJ energy per pulse. Orthogonal injection of ions from the quadrupole into the TOF section normally produce a mass resolving power 10,000 FWHM and accuracy within a few mDa in the TOF spectra in both MS and MS/MS modes, as long as the ion peak is reasonably intense. MS and MS/MS peak list were submitted to Profound and searched against the non-redundant NCBI human database using a mass accuracy of 20 ppm of monoisotopic peaks. Partial methionine oxidation and one trypsin miscleavage was allowed.
CMV-viremia was measured on peripheral blood buffy coat specimens using a semi-quantitative PCR assay developed at the Manitoba Cadham Provincial Laboratory that is accredited by the College of American Pathologists.
JMP IN software version 4.0.4 (SAS Institute Inc., Cary, N.C.) was used for statistical analysis. For categorical data, Fisher's exact test or Pearson's chi-square test was used. Parametric continuous data was analyzed by Student t-tests or one-way analysis of variance. For nonparametric continuous data, Wilcoxon or Kruskal-Wallis rank sum tests were used. A P-value <0.05 (two-sided test) was considered to indicate statistical significance.
Healthy people secrete less than 150 mg of protein in urine each day. Depending on the kidney or urinary tract system disease, proteinuria can reach more than 10 g per day. Basically, there are four different pathophysiological pathways that influence the protein content and composition of urine.
[I] Filtration from serum: The major part of urine proteins is derived from serum by filtration through the glomerular barrier. The glomerular barrier consists of the fenestrated endothelial cells, the glomerular basement membrane and the slit-diaphragm of the podocytes. The latter is considered to be predominantly responsible for the characteristics of the barrier. Proteins are thought to be retained from filtration into the urine based on their molecular weight, size, shape and net charge (75). Normally, proteins below 20 kDa are completely filtrated into urine, whereas larger proteins are generally retained in the serum. Albumin (66 kDa), for instance, would still pass the glomerular barrier based on its size, but it is speculated that its negative charge prevents filtration of large amounts. However, not everyone is in agreement with this hypothesis of charge selectivity (76).
[II] Tubular reabsorption and regurgitation: Many filtrated proteins bind to more or less specific receptors mainly on proximal tubular epithelial cells (e.g. megalin and cubilin). After binding, ligands are trafficked to lysosomes for degradation or endocytic vesicles for transcytosis back to the blood stream (77). Lysosomal degraded proteins may be directed back to the blood stream, but they are also regurgitated into the tubular lumen and ultimately excreted. The latter pathway was not recognized until recently and may have been underestimated (76,78). It is critical to take this pathway into account for proteomic analysis in urine, because not only intact proteins but also fragments of the same protein may be detectable.
[III] Active secretion: Some proteins are produced and secreted from tubular cells into the urine by an active process (e.g. Tamm-Horsfall protein) (79). Even whole vesicles can be released. Furthermore, cells with access to the urinary tract system can secrete proteins into it (e.g. neutrophils secrete α-defensins).
[IV] Cell-death derived proteins: Tubular cells undergo constant renewal and ‘old’/apoptotic cells are shed into the urine. Prescott estimated that, under physiological conditions, almost 2,000,000 tubular epithelial cells are sloughed into the urine each day (80). In addition, red and white blood cells as well as urothelial cells can be present in urine in significant amounts. Cells may stay intact or their membranes may rupture, releasing intracellular proteins into the urine.
The decoding of the human genome, developments in microtechnology, bioinformatics and mass spectrometry made it possible to investigate complex biological processes on a broad gene and protein level. Gene-microarrays (38,65) and MS-based proteomics (66,67) have gained widespread applications in biomedical research, including identification of candidate genes/proteins for diagnostic, prognostic and therapeutic purposes. However, both approaches have their limitations, which are mainly related to the technology itself (Table 1).
At present, there are several techniques to identify and compare the expression of proteins, each with advantages and disadvantages (Table 2). The most established method is protein separation by two-dimensional gel-electrophoresis (2-DE) followed by in-gel digestion and peptide mass fingerprinting by mass spectrometry. This method allows for the comparison of the relative abundance of proteins. However, there are several limitations of 2-DE as a separation method for proteomic studies. The resolvable range of molecular weights is limited at both ends, with a bias toward high abundance proteins. In addition, the technique requires relatively large amount of sample, is labour-intensive, and good gel-to-gel reproducibility can be hard to achieve (68,69). Thus, this approach is not optimal for high-throughput profiling.
An alternative approach uses one- or two-dimensional liquid chromatography as the separation step upstream from the mass spectrometer (liquid chromatography coupled to mass spectrometry, μLC-MS). While this technique provides information about the protein content of the samples, little information about their relative abundance can be obtained, unless the proteins/peptides are labelled first by isotope-coded affinity tags (70,71) or other protein/peptide labelling techniques (e.g. digestion with H2160 and H2180 mixture (72,73,74)). Furthermore, this method is still labour-intensive and has limited throughput. Surface-enhanced laser desorption/ionization time-of-flight mass-spectrometry (SELDI-TOF-MS) addresses some of the limitations of both 2-DE and μLC-MS. It combines matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to surface chromatography.
Specifically, a sample is applied to a chip surface carrying a functional group (e.g. hydrophobic, anion-exchange, cation-exchange, normal phase and metal-affinity). After incubation, proteins that do not bind to the surface are removed by a simple wash step, and bound proteins are analysed by mass spectrometry. This approach, in contrast to the others described, allows for high-throughput profiling of many clinical samples, but has limited sensitivity, resolution and mass accuracy.
SELDI-TOF-MS offers many advantages for protein profiling in urine. First, only 5 to 10 μL of sample is needed for one analysis. Second, due to the simple chip preparation, many samples can be analyzed quickly. Third, the washing step removes most of the salts, which otherwise interfere with mass spectrometric analysis. And fourth, the impact of different chromatographic chemistries can be analyzed, which may allow one to find optimal purification conditions for a protein of interest in a short time with small amounts of sample. However, sensitivity is moderate (especially in a complex mixture), and resolution above 25 kDa is low, resulting in a limited part of the urine proteome been detectable by SELDI-TOF-MS. In addition, standardization of analysis conditions is essential, and both extrinsic and intrinsic factors must be taken into account for accurate data interpretation.
Finally, protein microarrays, consisting of thousands of protein-specific capturing molecules (e.g. antibodies) in analogy to gene-microarrays, may revolutionize protein expression profiling. However, the few currently available antibodies largely limit this technology.
Urine Protein Profiling with SELDI-TOF-MS
In order to be able to compare the proteome of many samples, a high-throughput platform is mandatory. SELDI-TOF-MS system is a high-throughput platform available. Reliable profiling of clinical samples, required the reproducibility and the limitations of the SELDI-TOF-MS platform to be determined. In addition, several intrinsic (e.g. urine concentration, cellular components) and extrinsic (e.g. stability of urine proteins, storage) factors of urine were studied to confidently attribute differences in protein composition in various disease states to the disease process itself and not to confounding factors.
Reproducibility was evaluated by applying one urine sample to 14 spots and reading the spots using the protocol described in Example 1. The total number of detected peaks with an S/N-ratio ≧3 was 25 peaks/spectrum (range 23-29). Fourteen peaks common to all spectra were selected and compared with regard to their peak intensity by calculating the coefficient of variation. They ranged from 8 to 30%, with the lowest variation seen in the high intensity peaks and the higher variation seen in lower intensity peaks (
The most important extrinsic factors that influence reproducibility and peak detection are the matrix composition and the instrument settings. Matrix allows for efficient ionization and vaporization of proteins (82). The most popular matrices for the SELDI-TOF-MS system are SPA and CHCA. Saturated SPA is preferable for looking at masses above 10-20 kDa, while 10-20% CHCA provides the best resolution for proteins/peptides up to about 5 kDa. For urine protein profiling from 2-25 kDa, more peaks and a higher degree of resolution were observed with 35% CHCA. Instrument settings such as detector sensitivity, detector voltage, and laser intensity have to be determined individually. The higher the detector sensitivity and voltage or the laser intensity, the better the detection of high mass proteins. This is accompanied by an increase in background noise, which limits detection of low intensity peaks. The impact of matrix on the urine protein profile was determined by comparing different dilutions of CHCA and SPA (20%, 35%, 50% and 100%) with the otherwise unchanged protocol stated above. In the range from 2-25 kDa, 22, 26, 19 and 16 peaks were detected using 20%, 35%, 50% and 100% CHCA, respectively. In contrast, 13, 19, 11 and 10 peaks were detected using 20%, 35%, 50% and 100% SPA. Peak intensity below 8-10 kDa was higher with CHCA, whereas SPA yielded higher peak intensities above 8-10 kDa (urine protein profiles not shown).
The impact of spot sampling protocols was determined by comparing three different spot sampling protocols with respect to peak detection in undiluted and diluted urine: protocol 1 (standard protocol; see reference 96); protocol 2 (standard protocol modified to sample on only 5 different positions for a total of 80 shots/sample); protocol 3 (standard protocol modified to use a higher detector sensitivity (10 instead of 6)). Protocol 1 detected 34 peaks in undiluted urine, whereas protocols 2 and 3 detected only 21 and 26 peaks, respectively. In diluted urine (urine creatinine 3.75 mmol/l) the peak counts were 20, 11 and 13, respectively (urine protein profiles not shown).
The number of positions sampled on a spot is an important parameter for optimal peak detection. Ideally, all proteins are distributed homogeneously on the chip and are crystallized homogeneously in the matrix. If so, one would expect to generate the same spectra at every position. From the three spot sampling protocols it is clear, that there are ‘hot positions’, where proteins are clustered on the spot leading to the detection of an abundance of peaks with a high intensity. Similarly, there are ‘cold positions’, where only few or even no peaks are detected. Unfortunately, ‘hot position’ sampling may not accurately profile low abundant proteins due to ion suppression that can occur due to high abundance proteins. Therefore, the most representative spectra for a given urine sample is achieved by sampling many different spot positions and combining the data. This is especially true for dilute urine samples.
If the SELDI-TOF-MS approach is to be used in the assessment of clinical samples, it is important to assess the stability of the urine proteins prior to analysis. Recent studies have found little or no changes in albumin, retinol-binding protein, N-acetyl glucosaminidase, IgG and kappa/lambda light chain concentrations after storage at room temperature, 4° C., −20° C. and −70° C. (83,84,85,86).
First-void and mid-stream urine samples from three females and three males were analyzed within 2 hours from the time of collection, after storage for three days at room temperature and after three days at 4° C. In all six samples, only minor differences in the mid-stream urine protein profiles could be detected. However, in three first-void urines (two female, one male), storage for three days at room temperature or at 4° C. changed the spectra. A series of new peaks in the low molecular weight range was detected (
Storage of the urine samples at −70° C. did not change the spectra compared to those obtained before freezing. Furthermore, almost the same spectra could be generated after four freeze-thaw cycles, however, a loss of peaks was observed after the fifth freeze-thaw cycle (
Mid-stream urine is the standard for almost all urine analysis. In a clinical setting, there are always urine samples that are not mid-stream urines. Therefore, knowing the variation in urine protein profiles that may occur between first-void and mid-stream urines is important. In all three urine samples from males, there are almost no differences between the protein profile of first-void and mid-stream urine (
Another confounding variable in urine proteomic analysis is the presence of blood in urine. It can be present in urine under normal conditions (e.g. menstruation) or in association with urogenital tract pathologies. To investigate the impact of blood on the normal urine profile (
Blood was observed to be a major confounding variable affecting the normal urine protein profile. Not only did new peaks appear (i.e. peaks consistent with the masses of hemoglobin and albumin), but many of the normal peaks observed became undetectable. This is likely due to ion suppression by the blood proteins. Notably, even with a dilution of 10 μL blood in 64 ml diluted urine (1:6400 dilution), the peaks consistent with hemoglobin remained dominant. Clearly, such contamination invalidates any interpretation of the urine protein profile. Although centrifugation of the urine sample removes RBC, contamination with serum proteins will still continue to confound the urine protein profile
A dilute urine sample may limit the ability to detect the normal urine protein profile. To address the issue of urine concentration, urine was sampled from a healthy male person with a body weight of 75 kg after 20 hrs of no fluid intake. The measured urine creatinine was 15 mmol/L and the total protein was 0.11 g/L. At another time point, the same individual was challenged with 4 L of fluid over 2 hrs, leading to dilute urine with a creatinine of 0.9 mmol/L and a total protein of 0.03 g/L. While the concentrated urine showed the normal peak profile (
Depending on fluid intake the kidneys can concentrate urine to an output as low as 0.5 L/day, or dilute urine to almost 20 L/day. Under normal conditions, about 1-2 L urine are excreted per day. In a very dilute urine sample (urine creatinine 0.9 mmol/L), most of the proteins could not be detected on a NP20-chip. The threshold for a stable urine protein profile on a NP20-chip was a urine output of 2 L/day. Because every ProteinChip type has different binding capacities, the detection threshold has to be determined for every chip type individually.
Peak height and area under the peak have been used to reflect protein abundance (89,91). To determine if either the spectral peak intensity or area provides a means for reliable protein quantification, serial dilution of a single protein (ubiquitin, 8565 Da) was performed. There was an excellent correlation between the amount of protein in the sample and peak intensity (r2=0.95) or the area under the peak (r2=0.98) in non-normalized spectra (
Referring now to
To illustrate the importance of knowing the detection threshold, an example with the chemokine IP-10 in urine is described. Normal values measured by ELISA are 1-20 ng/L; during allograft rejection concentrations up to 1 μg/L have been reported (60). Even the later concentrations are 100-1000 times below the anticipated detection threshold of 100 μg-1 mg/L based on the experimental evidence from proteins in the same molecular weight range (ubiquitin and β2-microglobulin).
The detection of a protein by SELDI-TOF-MS is critically determined by its concentration in the sample, its binding to the chromatographic surface and its ionization process within the mass spectrometer. For single proteins, the detection threshold for α-defensins (3371 Da) was 10-100 ng/L (89), for ubiquitin (8565 Da) was 100 ng/L to 1 μg/L, and for albumin (66500 Da) was 1-6 mg/L, respectively. The increased detection threshold for high molecular weight proteins is well known and thought to be related to inferior ionization of large proteins. In a complex protein mixture (e.g. urine, serum), however, the detection threshold increases by roughly 10-1000 fold compared to the detection threshold for single proteins. This decrease in sensitivity is mainly caused by competition for binding sites (i.e. binding competition) on the ProteinChips and competition for ionization (i.e. ion suppression). Whereas the former is distinct to the SELDI-TOF-MS platform, the later is a common problem for all mass spectrometers. By changing the conditions for protein binding to different chromatographic surfaces, some proteins may be selected and enriched, whereas others may be excluded, allowing the detection limit to drop. However, the detection limit might be at best 10 times above the detection threshold for a single protein. Based on these experiments, the potentially detectable urine proteins by SELDI-TOF-MS can approximately be defined by their concentration and their molecular weight (
From July 1997 to March 2003, 2400 serial mid-stream urine samples from 212 renal transplant patients were collected. These 212 patients underwent a total of 693 protocol or clinically indicated core needle allograft biopsies. Based on allograft function, the clinical course and the allograft biopsy result, four rigidly defined patient groups were extracted from the whole patient population (n=212) as follows:
[1] Stable transplant group: Consists of 22 mid-stream urine samples (from 22 patients) obtained immediately before a protocol renal allograft biopsy performed within the first 12 months post-transplant. None of these patients had experienced DGF. All had stable allograft function (i.e. serum creatinine within 110% of baseline value at the time of biopsy), and none experienced a clinical or protocol biopsy-proven rejection prior to the date of examination. All biopsies met the criteria for adequacy and all were required to have an acute and chronic Banff score of zero (i.e. ai0t0v0g0 and ci0t0v0g0).
[2] Acute clinical rejection group: Consists of 18 mid-stream urine samples (from 18 patients) obtained immediately before a renal allograft biopsy performed within the first 12 months posttransplant. All experienced an elevation in creatinine >110% from baseline and the diagnosis of acute rejection required an acute Banff score ≧ai2t2v0g0. Patients with a chronic Banff score >ci1t1v0g0 were excluded in order to avoid chronic allograft nephropathy as a confounding variable in the analysis.
[3] Acute tubular necrosis (ATN) group: Consists of 5 mid-stream urine samples (from 5 patients) obtained immediately before a renal allograft biopsy performed within the first 6 days posttransplant to diagnose the cause of delayed graft function (DGF), which was defined as the need for hemodialysis within the first week or a drop of serum creatinine <50% from pre-transplant levels by day 5 post-transplant. Antibody mediated rejection was excluded based on a negative flow-crossmatch, and histological changes on the biopsy consistent with ATN. In all biopsies, the acute Banff score was ai0t0v0g0 and significant donor pathology was excluded by requiring a chronic Banff score of ≧ci1t1v0g0.
[4] Recurrent (or de novo) glomerulopathy group: Consists of 5 mid-stream urine samples (from 5 patients) obtained immediately before a renal allograft biopsy performed to diagnose the cause of proteinuria (≧1.5 g/day). The patients had diagnoses of membranous glomerulonephritis (GN), focalsegmental glomerulosclerosis or IgA-nephropathy and all had acute Banff scores ≦ai1t1v0g0. The acute clinical rejection group had more HLA-mismatches and a higher mean serum creatinine level at the time of the renal allograft biopsy compared to the stable transplant group. Otherwise, there were no significant differences between these groups (Table 4).
Normal control group: Consists of 28 mid-stream urine samples from 28 healthy individuals (14 female and 14 male, age 20-50 years).
Urinary tract infection (UTI) group: Consists of 5 mid-stream urine samples from 5 females obtained during an episode of a lower UTI, which was defined as requiring the clinical symptoms of a UTI, a leukocyte count in the urine sediment >40/high power field and a positive bacterial culture (>108 colony forming units).
Characterization of Urine Protein Profiles Associated with Individual Patient Groups
It was necessary to determine the urine protein profile of a ‘normal’ kidney transplant, and this was done by selecting urines from patients with immediate and persistent good graft function that had normal graft histology on protocol biopsy. This stringently defined control group is distinct as it includes histology; other groups attempting similar studies have inferred normal histology from a stable serum creatinine (57,56,97).
In the m/z range from 5000 to 12000 two distinct urine protein patterns were observed when comparing the normal control group or stable transplant group to the acute clinical rejection group. One urine protein profile (rejection pattern) had prominent peak clusters in three regions corresponding to m/z values of 5270-5550 (Region I; 5 peaks), 7050-7360 (Region II; 3 peaks), and 10530-11100 (Region III; 5 peaks) that always occurred together, whereas the other urine protein profile (normal pattern) had no peak clusters in these m/z regions (
Adherence to this stringent definition of ‘normal’ demonstrates that the urine protein profile from 18 of 22 patients (82%) in the stable transplant group was similar to the urine profile of normal non-transplanted individuals. The reliable identification of the urine protein pattern of the normal kidney transplant allowed for the clear differentiation, on visual inspection alone, of a distinct urine protein profile in the group with acute rejection (
The urine protein profile in the ATN and glomerulopathy groups did not show the pattern of rejection. Both ATN and glomerulopathies are important in the differential diagnosis of allograft dysfunction, and may represent pathophysiological models of allograft injury distinct from that due to the alloimmune response. Whereas ATN can be regarded as a model of injury to the tubules due to ischemia-reperfusion, in the glomerulopathies, the injury, although presumably immune in nature, is largely centered on the glomerular capillary. As these two pathological states did not show the characteristic pattern of rejection, the inventors infer that the urine proteins detected in acute rejection are related to recipient immune cells infiltrating the graft and/or to tubular epithelial cells that are involved in the allo-directed inflammation. It is acknowledged, however, that the possibility that the urine proteins associated with rejection may also be found in other causes of tubular-based pathology (i.e. calcineurin-inhibitor-toxicity, polyomavirus type BK-nephropathy, pyelonephritis) cannot be excluded. These latter outcomes are of relatively lower frequency in the patient population of the present study, such that it was not possible to generate pure examples of each in sufficient number to make any reliable conclusions. Indeed, it is notable, that in this patient population (n=212) only one patient (0.5%) developed polyomavirus type BK-nephropathy, which is a much lower incidence than reported from another centre (8%) (10).
Twenty-seven of 40 patients (68%) in the stable transplant and acute clinical rejection groups were tested for the presence of CMV-viremia at the time of renal allograft biopsy. Five patients tested positive; however none had or developed CMV-disease subsequently. CMV-viremia was found in 2 of 21 patients (10%) with the rejection pattern and in 3 of 19 patients (16%) with the normal pattern (P=0.83) (Table 5). No additional peaks in the urine protein profiles from patients who had CMV-viremia were detected.
An additional potential confounder of the diagnostic specificity of the urine protein profile observed in allograft rejection is systemic inflammation that could lead to the filtration of inflammatory proteins (e.g. chemokines, cytokines) by the transplant kidney. Post-transplant CMV viremia, which has a high incidence in kidney transplant recipients (101,102) but very rarely infects the allograft (103,104), is one of the most common causes of systemic inflammation post-transplant.
Indeed, the inventors have previously reported that CMV-viremia is a significant confounding variable when examining activated T-cells in the circulation as a possible non-invasive correlate of biopsy proven allograft rejection (61). In the current study, no correlation was found between CMV-viremia and the urine profile of rejection, which argues against systemic inflammation associated with CMV viremia as a significant confounding factor. While this does not rule out the possibility that other systemic inflammatory processes may mimic the urine profile seen in allograft rejection, it suggests that this is probably less likely.
To further determine the specificity of the normal and rejection pattern, serial urine protein profiles in the stable transplant and acute clinical rejection groups were examined and correlated with the clinico-pathological course of the renal allograft. In particular, four specific outcomes were of interest: [1] the stable course persisted; [2] the stable transplant patient subsequently had an acute clinical rejection; [3] acute clinical rejection resolved to a stable course; [4] acute clinical rejection recurred.
In the stable transplant group, there were sufficient urine and histology samples for sequential analysis to evaluate 12 of the 18 patients that originally had a normal pattern (
In the acute clinical rejection group, there were sufficient urine and histology samples for sequential analysis to evaluate 12 of the 17 patients that originally had a rejection pattern (
One patient had two subsequent normal protocol biopsies, but the creatinine remained elevated at the level seen during the acute rejection episode (20% above baseline) and the urine always showed the rejection pattern. In 6 patients the allograft function returned to baseline and subsequent protocol biopsies were interpreted as normal (n=3) or borderline rejection (n=3). One patient had acute clinical rejection (Banff IB) on week 7 post-transplant. After treatment with high dose oral steroids the serum creatinine normalized and remained stable. Subsequent allograft biopsies were normal. The urine protein profile showed the normal pattern 3 week prior to the rejection episode, changed to the rejection pattern at the time of rejection, and returned to the normal pattern consistent with the subsequent allograft biopsies and the allograft function. All urine samples from these patients changed to the normal pattern (
It was of interest that the protein profile of rejection was similar regardless of the histological severity (Banff IA vs. IB) or type (Banff IA/B vs. IIA). This finding might represent a relative limitation of the technique of urine proteomics in identifying biomarkers specific for tubulo-interstitial versus vascular rejection. However, because the assignment of histological severity/type of acute rejection is based upon a small biopsy sample of a large organ, urine profiling, which is representative of the entire allograft, may be pointing to the extent of heterogeneity of inflammation within the allograft, a fact that renal transplant pathologists are well aware of (49). The correlation between the changes in serial urine profiles and the clinico-pathological course of the patients provided additional support that the detected proteins are related to acute allograft rejection.
Although there were significant differences in the urine profiles between the stable transplant and the acute clinical rejection groups, there were also one ‘false negative’ and four ‘false positives’ samples. The only patient with the ‘false negative’ urine profile in the acute clinical rejection group had no specific clinical or demographic feature. That patient had a course of a subclinical rejection (ai3t3g0v0) followed by a clinical rejection (ai3t3g0v1)—both treated with oral high dose steroids—and returned to normal histology (ai0t1g0v0) 15 weeks later. The inventors found no obvious explanation for this ‘false negative’ result. Theoretically, a low protein concentration in dilute urine may influence the ability to detect a rejection pattern. However, the protein concentration of the urine samples from the stable transplant and the acute clinical rejection group were similar, making inadequate protein load an unlikely explanation for the absence of the rejection pattern. The four patients with ‘false positive’ urine profiles in the stable transplant group also had no specific clinical or demographic features at the time of the biopsy. However, one of them went on to subclinical rejection (ai1t3g0v0) 9 weeks later and one experienced an acute clinical rejection and polyomavirus type BK-nephropathy (BK-NP) 13 weeks later. The other two patients had a normal transplant course with stable graft function. There are mainly two possible explanations for these ‘false positive’ results. First, they are true ‘false positives’ and cannot be explained. Second, they are not ‘false positives’ as the urine profile may be detecting an early rejection process that was missed by the allograft biopsy (i.e. sampling error) (100,49).
As the urine samples from the transplanted patients were stored non-centrifuged at −80 C, subsequent cell lysis due to freeze-thawing, the analysed urine samples will contain intracellular proteins from cells present in the urine. To investigate whether the release of intracellular proteins of red blood cells (RBC), leucocytes and tubular epithelial cells due to freeze-thawing is responsible for generating the rejection pattern, the inventors compared an ‘acute clinical rejection’ urine sample frozen with and without pre-centrifugation (
Urine profiles of the various groups could have been altered by the procedures of urine collection and storage. Due to the fact that all urine samples were stored non-centrifuged, the rejection pattern may have derived from intracellular proteins of leucocytes, RBC or tubular epithelial cells released after a freeze-thaw cycle. Interestingly, in one of the rejection cases the inventors found that lysis of RBC prevented the detection of the rejection pattern due to ion suppression. However, precentrifugation to remove the RBC prior to freeze-thawing of this sample allowed the rejection pattern to be detected. Therefore, this argues that the pattern is not necessarily derived from cell lysis associated with a freeze-thaw cycle.
The inventors first determined the pI of the rejection pattern proteins in order to subsequently use an extraction method (i.e. ion-exchange beads) as an initial step to concentrate the target proteins. With the use of cation- and anion-exchange beads the pI was estimated to be around 7.0 (
After in-solution digestion, the target proteins were identified by μLCMS and liquid chromatography coupled to tandem mass spectrometry (μLC-MS/MS) as a cleaved form of β2-microglobulin. As all the protein(s) responsible for the peak clusters remained in one single fraction after two purification steps involving cation-exchange and reverse-phase chromatography, a close relationship between each of the peaks seemed obvious. In fact, analysis of the purified and trypsin-digested sample by μLC-MALDI-MS (/MS) revealed that all peptides found belong to one protein, namely β2-microglobulin. Complete decoupling of μLC and MALDI-MS (/MS) techniques enables detailed analysis of the deposited sample without any time constraints. This feature was used to find small and/or low-abundant peptides by single MS mass measurement and confirming their sequence by MS/MS, resulting in almost complete coverage of the 99 amino acid long β2-microglobulin sequence, with the exception of a five amino acid long peptide (L64-T68) (
[I]I1QRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERI EKVEHSDLSFSKDWS61 (SEQ ID NO:4) with a predicted molecular weight of 7047.83 Da (SELDI-TOF-MS mass 7042.9 Da (−4.9 Da));
[II] E69FTPTEKDEYACRVNHVTLSQPKIVKWDRDM99 (SEQ ID NO: 5) with a predicted molecular weight of 3737.22 Da (SELDI-TOF-MS mass 3733.0 Da (−4.2 Da)); and
[III] F70TPTEKDEYACRVNHVTLSQPKIVKWDRDM99 (SEQ ID NO:6) with a predicted molecular weight of 3608.10 Da (SELDI-TOF-MS mass 3603.6 Da (−4.5 Da))
Therefore, these three cleavage sites combined with or without the theoretical removal of the disulphide bond explain five of the seven major peaks detected by SELDI-TOF-MS (
However, the initial described rejection pattern had prominent peak clusters at 5.27-5.55 kDa (5 peaks), 7.05-7.36 kDa (3 peaks), and 10.53-11.1 kDa (5 peaks). The unaccounted SELDI-TOF-MS peaks at 7.2 kD and 7.36 kD, as well as the concomitant appearing or disappearing peaks at 10.95 kD and 11.1 kD (double charged ions at 5.48 kDa and 5.55 kDa) can be explained by a different initial cleavage site (Y63 instead of S61) with subsequent partial removing of Y63 and F62 (
The two last remaining unaccounted peaks of the original rejection pattern (5.27 kDa and 10.53 kDa) can most likely be explained by removing F70, which was an observed cleavage site (
Proteins can be separated based on [I] their molecular weight, [II] their pI and [III] their, hydrophobicity. The use of ion-exchange beads as a first step to purify the target proteins with a pI of 7.0 from urine offered two advantages. First, it allows one to concentrate the target proteins, and second, many proteins with lower pIs could be excluded. Subsequently, the high-resolution ability of RP-HPLC allowed purifying the cleaved β2-microglobulin. Indeed, it was even possible to separate the cleaved form (eluted at around 31% acetonitrile) from the intact form (eluted at around 33% acetonitrile), which only differ by seven amino acids. Identification of cleaved β2-microglobulin by μLC-MS and μLC-MS/MS is very reliable. Not only were all the peptides corresponding to the β2-microglobulin sequence (without the cleaved piece ‘F62YLLYYT68’ (SEQ ID NO:10)) found and confirmed by MS/MS, but the observed and predicted cleaved forms could explain 11 of 13 peaks of the rejection pattern detected by SELDI-TOF-MS. However, the question remains, why cleaved β2-microglobulin produces the observed multiple peaks on the SELDI-TOF-MS spectra. β2-microglobulin consists of 99 amino acids and contains one disulphide bond (C25-C80). Purified human β2-microglobulin from urine is not fragmented when analysed by SELDI-TOF-MS and only the double charged species is observed beside the parent ion (
Based on the SELDI-TOF-MS detected β2-microglobulin fragments, the initial non-tryptic cleavage sites were postulated to be Y63 and T68. Thereafter additional major cleavages occur at S61, F62 and E69, resulting in 11 of 13 peaks contributing to the rejection pattern (
β2-microglobulin is freely filtered through the glomerular barrier and is normally reabsorbed by proximal tubular epithelial cells to a large extent. Therefore, changes in β2-microglobulin metabolism and excretion are mainly dependent on the function of the tubular epithelial cells. In addition, proteinases in urine may mostly be derived from these cells (107,108,109). Taken together, the presence of cleaved β2-microglobulin in urine is most likely to be associated with tubular epithelial cell stress/injury. Interestingly, in patients with pure humoral rejection (n=3, data not shown), which does not target the tubular cells, cleaved β2-microglobulin was not detectable by SELDI-TOF-MS further supporting the association between tubular cell stress/injury and the presence of cleaved β2-microglobulin. Whether cleaved β2-microglobulin is specific for tubular cell stress/injury due to rejection is not known yet and needs to be addressed in further analysis of samples with different pathologies affecting the tubuli (i.e. CNI-toxicity, polyomavirus type BK-nephropathy, pyelonephritis).
Alternatively to the hypothesis that urinary cleaved β2-microglobulin is derived from filtration of intact recipient β2-microglobulin with subsequent intracellular or intraluminal fragmentation in the allograft, it could also be derived directly from kidney donor allograft cells (e.g. tubular epithelial cells) or from recipient immune system cells in the allograft (e.g. CTL, macrophages).
Other approaches can be used to profile different subsets of urine proteins for their potential as biomarkers for renal allograft rejection. Such approaches include comparative analysis of urine samples from stable transplants and patients undergoing rejection (i.e. differential protein profiling).
The Aebersold laboratory introduced the concept of isotope coded affinity tags (ICAT), in an effort to provide a means for direct comparison of protein levels in two samples by mass spectrometry (70). The ICAT reagent is in two structurally identical forms, which only differ by the presence of heavy, H, or light, L, species of stable isotopes of carbon or hydrogen (i.e. C12/C13 or H1/H2) resulting in H and L forms of ICAT. Equal amounts of the two samples to be compared are reduced, labeled separately, each with only one species of ICAT, and then pooled for processing. ICAT reacts with free —SH groups and introduces a selectable biotin affinity tag which allows for the isolation of the tag labelled peptides from the overall digest. This step was designed to reduce the overall complexity of the samples in the subsequent chromatographic and mass spectrometric steps. The tagged peptides can be separated by 1 or 2 dimensional μLC on-line with a mass spectrometer. In single MS mode peptides from the same protein species but labelled with the heavy and light forms of the tag will display a predictable separation in m/z depending on the charge state of the peptides. Integration of the areas of the isotope cluster for the H and L species provides a basis for comparing their relative abundance.
Subsequent analysis of the parent ion by tandem MS provides protein identification. Thus in a single experiment it is possible to obtain information on relative protein abundance and identity of the altered expression patterns. The limitations of the approach relate to the relatively narrow dynamic range (i.e. ˜10 fold) and the requirement for cysteines in the proteins.
Another approach for differential protein profiling employs digestion in the presence of O16 or O18 (110). Equal quantities of urinary proteins from the groups to be compared will be dialysed and lyophilised. The samples will then be resuspended in buffer containing exclusively either O16 or O18 H2O. During trypsin digestion peptide bonds C terminal to the basic residues are hydrolysed resulting in the incorporation of OH into the C terminal carboxyl group. Thus by digesting the two protein mixtures to be compared in different forms of H2O sets of peptides differing by 2 mass units are generated. Combining equal quantities of the protein digests and fractionating in a similar fashion to the ICAT provides a means of performing a similar type of quantitative comparison and protein identification. This scheme labels all peptides and is only dependent on the presence of cleavage sites for trypsin rather than cysteine.
In summary, the results from the ICAT and the hydrolytic labelling offer the means to obtain broad comparative analysis of the urine samples of interest. However, both methods do not allow for high throughput analysis making the selection of few clinically well defined samples mandatory to allow meaningful interpretation.
A urine from a patient, which showed in the SELDI-TOF-MS spectrum both the intact and the cleaved form of β2-microglobulin, was brought to pH 3, 4.5, 6 and 8. After 6 to 24 hours the intact form of β2-microglobulin was not detectable anymore in urines with pH 3 and 4.5, whereas it was detectable in unchanged intensity in urines with pH 6 and 8.
[ii] Inhibition of Initial Cleavage of Intact Urinary β2-Microglobulin by Pepstatin
Pepstatin is a well-established inhibitor of aspartic proteinases. A urine from a patient, which showed in the SELDI-TOF-MS spectrum both the intact and the cleaved form of β2-microglobulin, was brought to pH 4.5 that the inventors demonstrated in [i] to be required for initial cleavage of intact urinary β2-microglobulin. Different proteinase-inhibitors were added (Pepstatin and Complete Mini EDTA-free, both from Roche, Switzerland). After 6 to 24 hours the intact form of β2-microglobulin was not detectable anymore in urine spiked with Complete Mini EDTA-free and in untreated urine, whereas it was still detectable in unchanged intensity in urines spikes with Pepstatin.
The initial cleavage of intact urinary β2-microglobulin is dependent on a pH<6 and can be inhibited by Pepstatin. This indicates that the initial cleavage is done by aspartic proteinase(s), which are known to be mainly active at lower pH. So far, two aspartic proteinases were found in renal tubular epithelial cells and in urine (i.e. cathepsin D and napsin A), and one or both may be responsible for the initial cleavage of intact urinary β2-microglobulin. Based on the preliminary data it appears that the initial cleavage sites are at Y63 and L65 (see
Cleavage of β2-microglobulin by aspartic proteinase(s) creates two chains that are still connected through the disulphide bond (C25-C80). The long chain of cleaved β2-microglobulin consists of 63 amino acids (I1-Y63), the short chain of 34 amino acids (Y66-M99).
Further confirmed non-tryptic cleavage sites on the long chain occur at F62 and then at S61, resulting in three major forms of long chains with calculated molecular weights of 7358.19 Da (I1-Y63), 7195.01 Da (I1-F62) and 7047.83 Da (I1-S61) (see
Many cleavage sites on the short chain have been confirmed by μLC-MS/MS (K75, E74, T73, P72, T71, F70 and E69). Only two of the resulting forms can be confidently found by SELDI-TOF-MS with calculated molecular weights of 3737.22 Da (E69-M99) and 3608.10 Da (F70-M99) (see
During analysis by SELDI-TOF-MS disulphide bonds can break resulting in the detection of the different forms of single short and long chains of cleaved β2-microglobulin. However, cleaved β2-microglobulin forms consisting of both chains, which are connected through the disulphide bond, are still present (see
One mL of a urine sample from a healthy individual (total protein 110 mg/L, creatinine 18.9 mM, pH 5) and 1 mL of a urine sample from a patient with an acute clinical rejection episode showing the protein peak clusters (total protein 230 mg/L, creatinine 11.6 mM, pH 5) were incubated for 16 hours at 37° C. to degrade existing intact and cleaved β2-m. Four hundred μL of each sample were mixed with sodium acetate pH 5 (final concentration 166 mM) to ensure, stable pH and divided into four portions of 100 μL. Pepstatin (final concentration 14.5 μM), Complete-Mini (EDTA-free) (final concentration 0.2 tablets/mL), EDTA (final concentration 20 mM) or no protease inhibitors were added to each portion. Another 100 μL of each urine sample was brought to pH 6 by adding MES pH 6 (final concentration 166 mM). Purified intact β2-microglobulin (final concentration 10 mg/L) was added to all portions. SELDI-TOF-MS analysis was performed immediately and after 1, 2, 4, 6 and 24 hours of incubation at 37° C.
The characteristic protein peak clusters of cleaved β2-microglobulin could be generated by spiking intact β2-microglobulin into even normal urine under specific conditions. Cleavage of intact β2-microglobulin was only observed at a urine pH<6 and could be inhibited by the aspartic protease inhibitor pepstatin, but not by cysteine & serine protease inhibitors (Complete-Mini (EDTA-free)) or a metalloprotease inhibitor (EDTA) (
One hundred μL of a urine sample from a healthy individual (important parameters stated above) and 100 μL of a urine sample from a patient with the protein peak clusters (important parameters stated above) were incubated for 16 hours at 37° C. to degrade existing intact and cleaved β2-microglobulin. Sodium acetate pH 5 (final concentration 166 mM) was added to ensure stable pH, and then purified intact β2-microglobulin (final concentration 10 mg/L) was spiked into each sample. SELDI-TOF-MS analysis was performed immediately and after 1, 2, 4, 6 and 24 hours of incubation at 37° C.
The generated cleaved β2-microglobulin forms were identical in urine samples from a healthy individual and a patient with an acute clinical rejection episode. However, the course of cleavage/degradation was much faster in the latter one, suggesting that more protease activity was present in the urine sample collected during acute clinical allograft rejection (
Cleaved β2-microglobulin forms representing the protein peak clusters are produced early in the degradation process of β2-microglobulin and are by far the most abundant cleaved β2-microglobulin forms based on peak intensity of the SELDI-TOF-MS spectra. Thereafter more cleavages occur (see
One mL of a urine sample from a healthy individual (important parameters stated above) was incubated for 16 hours at 37° C. to degrade existing intact and cleaved β2-microglobulin. Sodium acetate pH 5 (final concentration 166 mM) was added to ensure stable pH and the sample was divided into nine portions of 100 μL each. Different amounts of purified intact β2-microglobulin (final concentrations from 0.05-10 mg/L) were spiked into these 100 μL portions. SELDI-TOF-MS analysis was performed immediately and after 6 hours of incubation at 37° C.
To determine the detection threshold of SELDI-TOF-MS for cleaved β2-microglobulin, we added different amounts of intact β2-microglobulin into a urine from a healthy individual and analysed the samples for the presence of cleaved β2-microglobulin forms after 6 hours of incubation at 37° C. The detection threshold of SELDI-TOF-MS for the cleaved β2-microglobulin was between 0.1 and 0.5 mg/L of added intact β2-microglobulin (
As stated above, urine pH is critical for generation of cleaved β2-microglobulin. Therefore, the inventors retrospectively analysed urine pH in all available samples (n=63) from the previous examples where the identified cleaved β2-microglobulin forms were closely associated with acute clinical allograft rejection (10). Urine pH in the acute clinical rejection group (n=18; pH=5.26±0.33, range 4.7-5.8) was significantly lower than in the stable transplant group (n=22; pH=5.58±0.50, range 5.0-6.5) (p=0.037) and the healthy control group (n=23; pH=5.89±0.56, range 5.0-7.0) (p=0.0004). All urine samples with cleaved β2-microglobulin forms detectable by SELDI-TOF-MS had a urine pH<6. However, 20 of the analysed 63 urine samples (1 in the acute clinical rejection group, 11 in the stable transplant group, and 8 in the healthy individuals group) had pH<6 without SELDI-TOF-MS-detectable cleaved β2-microglobulin forms. Based on the experiment presented above (
Cleaved urinary β2-microglobulin can be regarded as a marker for tubular cell stress/injury, because all patients in the acute clinical allograft rejection group had at least mild tubulitis (i.e. Banff acute Score ≧i2t2). As was demonstrated, tubular cell stress/injury during allograft rejection can lead to (i) decreased reabsorption of intact β2-microglobulin, (ii) increased amounts of proteases in urine, and (iii) lower urine pH (
Therefore, cleaved urinary β2-microglobulin represents several pathophysiological processes occurring during tubular cell stress/injury related to tubulointerstitial allograft rejection. However, it is not believed that cleaved urinary β2-microglobulin is specific for tubulointerstitial allograft rejection, but may be a sensitive marker for any kind of tubular cell stress/injury (e.g. CI-nephrotoxicity, polyomavirus type BK-nephropathy).
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
aApplies to tubules no more than mildly atrophic
aIn the most severely affected vessel. Note if lesions characteristic
a)P = 0.003 vs. stable transplant group
b)P < 0.001 vs. stable transplant group
c)P = 0.14 vs. acute clinical rejection group. P < 0.001 vs, recurrent or de novo glomerulopathy group
d)Not included for statistical analysis
e)Not included for statistical comparison (3 of 5 patients were on hemodialysis)
a) Consists of 18 patients from the stable transplant group plus 1 patient from the acute clinical rejection group
b) Consists of 4 patients from the stable transplant group plus 17 patients from the acute clinical rejection group
c) CMV-PCR was not performed for the following reasons: CMV sero-negativity of both donor and recipient (n = 2); test was not ordered (n = 3); or only CMV pp65-antigen was evaluated (n = 1; patient tested negative)
d) CMV-PCR was not performed for the following reasons: CMV sero-negativity of both donor and recipient (n = 3); test was not ordered (n = 3); or only CMV pp65-antigen was evaluated (n = 1; patient tested negative)
NGER
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2005/001423 | 9/20/2005 | WO | 00 | 10/20/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60611291 | Sep 2004 | US |
